Recent evidence demonstrates an essential role for cellular cathepsins in viral glycoprotein processing and cellular entry for the highly pathogenic viruses Ebola, SARS coronavirus and Nipah/Hendra. Cathepsin L appears to be important for Nipah and SARS coronavirus virus glycoprotein activation while Ebola requires both cathepsins L and B for viral entry. Inhibitors of these enzymes effectively block viral entry and replication in cell culture. Studies in mice with broad spectrum cathepsin inhibitors as well as specific inhibitors and genetic knockouts all suggest that cathepsin activity can be impaired in vivo without significant detrimental effects. In preliminary studies we have used a novel methodology to screen chemical libraries on microarrays and have identified two previously unrecognized cathepsin L inhibitors. One of these inhibitors, PC-185, is highly specific for cathepsin L and blocks Ebola entry with an IC50 of ~193nM. This proposal will build upon these significant preliminary findings and will develop cathepsin inhibitors as therapeutics for Ebola. For this application, we propose the following aims: Specific Aim 1) Utilize high throughput screening of diverse libraries to identify additional new cathepsin inhibitors. Analyze structureactivity relationships (SAR), optimize lead compounds, and develop second-generation agents and screening libraries. Specific Aim 2) Test the candidate compounds for inhibition of viral entry using a rapid, quantitative and safe assay employing viral pseudotypes carrying the glycoproteins of Ebola virus. A novel virus-like particle system that we developed to study Ebola entry will be employed to confirm the ability of lead compounds to block filamentous Ebola infection. Additionally, we will send promising candidates to collaborators at USAMRIID for efficacy testing against Ebola infection. Effective candidate compounds will be cross screened against SARS-CoV and Hendra infections. Specific Aim 3) Determine the mechanism(s) by which viruses can escape cathepsin inhibition by selection of escape mutants using a novel VSV vector that relies upon Ebola GP for replication (VSV-GP).